NO Knocks Out Other Germs

Nitric oxide’s reputation as a vital biological molecule has been
bolstered by other recent work conducted at Brookhaven. In collaboration
with The Johns Hopkins University, a Brookhaven team has shown that human
adenovirus treated with nitric oxide (NO) showed a dramatic decrease in
infectivity. More...

Nitric Oxide Chemistry Contributes To Cystic Fibrosis Research

Because of Lymar’s expertise in inorganic nitrogen chemistry, Hassett’s
team sought his assistance in their observation of the bactericidal
action of sodium nitrite on the mucoid forms of Pseudomonas aeruginosa.

Chemist Sergei Lymar

“I felt that they were up to something potentially very important,”
Lymar said. “It has quickly become clear that the species toxic to these
bacteria is not the nitrite itself, but one of the downstream products
of its decomposition in a slightly acidic environment. This chemistry is
complex, including a peculiar mix of very rapid and slow reactions and a
number of products and intermediates, so we had to resort to computer
simulations to understand what is happening on the relatively long time
scales typical of bactericidal assays.”

The project was quite a change of pace for Lymar, who is used to
dealing with reactions occurring in microseconds. In this case,
computations revealed that the reactions take hours and days to unfold.
His modeling clearly pointed to nitric oxide as the most probable toxic
species and also helped to design experiments that could test this and
other model predictions.

“We knew from experience, that this kind of experiment is very
susceptible to artifacts,” Lymar said. “One needs to work cleanly,
quantitatively, with the right mixtures of gases -- and completely
eliminate contact with air. When all that was done, the model
predictions checked out beautifully.”

"For the mucoid bacteria, nitric oxide can be a messenger of death."-- Sergei Lymar

Both simulations and experiments showed highly desirable properties
of acidified sodium nitrite as a time-release capsule drug-delivery
system. In a matter of hours, it elevated the nitric oxide content of
cell cultures and then maintained these levels over days, constantly
exposing bacteria to this active ingredient and killing them. Similar
rates of killing were observed in a series of experiments designed to
produce the same nitric oxide (NO) levels either by various combinations
of nitrite concentration and acidity or by applying nitric oxide-argon
gas mixtures in the absence of nitrite. Another remarkable feature of
the acidified nitrite chemistry is a built-in “feedback mechanism” where
the system will increase the rate of nitric oxide production in response
to an attempt by bacteria to get rid of NO.

“Although we are convinced that the bactericidal action of acidic
nitrite is due to nitric oxide that it generates, we are less certain
about the molecular mechanism of its toxicity,” Lymar said.

One important clue might be in the dramatic decline of the bacterial
population upon exposure to NO. One day after treatment, the organism
population decreased by roughly 90 percent; the next day, 90 percent of
what remained was eliminated.

“This means that treatment wouldn’t need to be long-term,” Lymar
said. “But what it also tells us is that nitric oxide might act as a
trigger of bacterial death. An organism that has survived to any given
time appears “to have no idea” that it has spent a day or two in a toxic
environment until something hits it. It is difficult to reconcile such
behavior with slowly accumulating damage to a microbe; it rather
suggests that only a small number of reactive events, or perhaps even a
single event, trigger the bacterial death.”

“This pattern is consistent with a well recognized role of nitric
oxide as a regulator in biology. It can trigger biochemical processes,
but doesn’t necessarily participate directly in them. For the mucoid
bacteria, nitric oxide can be a messenger of death.”

The research was funded by the National Institutes of Health and the
Cystic Fibrosis Foundation and Lymar’s work on nitrogen oxides is
supported by the U.S. Department of Energy.

One of ten national laboratories overseen and primarily
funded by the Office of Science of the U.S. Department of Energy (DOE),
Brookhaven National Laboratory conducts research in the physical,
biomedical, and environmental sciences, as well as in energy technologies
and national security. Brookhaven Lab also builds and operates major
scientific facilities available to university, industry and government
researchers. Brookhaven is operated and managed for DOE’s Office of Science
by Brookhaven Science Associates, a limited-liability company founded by
Stony Brook University, the largest academic user of Laboratory facilities,
and Battelle, a nonprofit, applied science and technology organization.